PROJECT SUMMARY The goal of this study is to investigate how the properties of proteins are impacted by post-translational modifications (PTMs) associated with protein prenylation, specifically the proteolytic and carboxylmethylation events that typically follow isoprenylation. Most studies to date have investigated this issue using a select few reporters, including the Ras GTPases and the yeast a-factor mating pheromone. Our studies are bringing a new understanding to this issue by promoting a general view that the PTMs occurring to prenylated proteins are not necessarily coupled and are key regulators of protein function and localization. We have proposed two aims. One will investigate the coupling of PTMs using a set of prenylated protein reporters outside those previously studied. Our studies will challenge the conventional paradigm for how prenylated proteins are modified, which in turn will provide guidance to strategies aimed at interfering with protein prenylation and related downstream events as disease therapies. The second aim will investigate the target specificities of the Rce1p and Ste24p proteases that act on prenylated proteins. Our studies will demonstrate that these proteases have very distinct enzymatic profiles, which will provide additional guidance on the specific targeting of these enzymes in disease therapies. We bring to bear on our investigations an exceptionally strong set of preliminary findings, the complementary expertise of several research groups, and a comprehensive molecular toolbox for the study of prenylated proteins and the proteases involved.